Inhalation of nebulised therapeutics has become a frequent drug delivery method for the treatment of lung diseases such as asthma or lung infections and other respiratory diseases. However, such mode of administration usually requires repeating the treatment several times per day, which however, may not be always compliant with patient's health conditions depending on the disease he suffers from or the grade of severity of the disease itself. Frequent inhalations of therapeutic agents also constitutes a severe life-limiting stress. Continuous nebulisation therapy of β2 agonists has proved to be a useful alternative therapy for patients suffering from severe asthma (Raabe O. G., et al., Ann. Allergy Asthma Immunol., 1998, 80, 499). However, even in this case, the therapy is very time consuming and renders the patient's life uncomfortable.
Serious lung diseases such as lung cancer or cystic fibrosis are anyway still largely treated by systemic therapies which are unfortunately associated to significant side effects.
Lung's anatomy and physiology are well adapted to process exogenous nebulised substances for clearance in order to protect the organ. It is also recognized that also in the case of volunteer treatment exposure through inhalation of therapeutic agents, the latter are fast cleared therefore influencing negatively and thereby limiting the benefit of inhalation therapy.
It is further well known that enzymes aimed at detoxifying organs from external aggression are also present in the lung. Some of those which play an important role in the lung belong to the super family of proteins (i.e., AKR). AKR and short-chain dehydrogenases/reductases (i.e., SDRs) are the main enzymes that catalyze oxidation-reduction reactions involving a xenobiotic carbonyl. Among SDR superfamily, carbonyl reductases (i.e., CBRs) exhibit broad substrate specificity for carbonyl bearing xenobiotics (Matsunaga T., et al., Drug Metab. Pharmacokinet., 2006, 21, 1, 1).
Attempts of local delivery of chemotherapeutics by aerosol have been recently reported in pre-clinical models of lung cancer showing reduced toxicity compared to systemic administration (Fulzele, S. V., et al., J. Pharm. Pharmacol., 2006, 58, 3, 327).
A dry powder formulation for inhalation of temozolomide has recently been reported. The latter required a particle size reduction to enable a 51% release of the administered dose (Wauthoz N., et al., Pharm. Res., 2011, 28, 762), and also the presence of biocompatible and biodegradable phospholipids as surfactants to stabilise the aqueous temozolomide suspension (Wauthoz N., et al., Eur. J. Pharm. Sci., 2010, 39, 402).
A Phase I clinical trial involving cisplatin was conducted positively in order to investigate the safety and pharmacokinetics of aerosolized Sustained Release Lipid Inhalation Targeting (SLIT) cisplatin in patients with lung carcinoma. However, still quite a lot of side effects (i.e., nausea, vomiting, dyspnea, fatigue and hoarseness) were encountered in this study (Wittgen B. P. H., et al., Clin. Cancer Res., 2007, 13, 2414).
Nebulisation of 5-fluorouracil in dogs demonstrated that the drug could reach very high concentrations mainly in the trachea, to a less extent but still important into bronchi and oesophagus and a reduced concentration (i.e., one fiftieth of the one encountered in the trachea) into the lymph nodes at the bronchial level (Tatsumura T., et al., Br. J. Cancer, 1993, 68, 1146).
Nevertheless, diffusion of these aerosolized small chemical drugs to the blood is still a relevant issue together with the need of repeated administration due to the short lung half life of these molecules.
Aerosol gene delivery is another application long pursued for the targeted therapy of lung diseases. After the cloning of the cystic fibrosis gene, there was great interest in the non-invasive delivery of genes directly to the pulmonary surfaces by aerosol. This approach could have application to inoperable pulmonary cancers as well and most early efforts focused mainly on the use of nonviral vectors, primarily cationic lipids and other formulation excipients (Densmore C. L., et al., J. Gene Med., 1999, 1, 4, 251; Densmore C. L., et al., Mol. Ther., 2000, 1, 2, 180).
Unfortunately, nebulisation shear forces, and inefficient pulmonary uptake and residence of aerosolized protein therapeutics coupled to low expression of genetic vectors have generally resulted in a poor therapeutic effect (Schwarz, L. A., et al., Hum. Gene Ther., 1996, 7, 731). Consequently, the interest in lung delivery of protein-containing biologically active derivatives by aerosol has diminished in recent years.
If inhalation of therapeutic proteins had been sought as an attractive solution for targeted therapy of the lung, the formulation of protein to be nebulised still remains challenging. Indeed, in order for the aerosolized substance to penetrate the lungs in-depth, formulation requiring scrupulous selection of additives and particle size (i.e., up to 3 μm) have to be adjusted meticulously (Choi W. S., et al., Proc. Natl. Acad. Sci., 98, 20, 11103). Furthermore, the quaternary, but also the secondary and tertiary structure of the protein can be altered by the nebulisation process. To overcome this drawback, Arakawa T., et al. disclosed the use of polyethylene glycol and/or a surfactant to preserve said structural conformation prior to nebulisation (WO199503034).
Variously grafted nanoparticles designed for aerosol administration to target the lungs and which can entrapped different anti-cancer drugs have also been described lately, but were reported to present some inflammatory disadvantages (Dailey L. A., et al., Toxicol. Appl. Pharmacol., 2006, 215, 1, 100).
A polymer-based nanoparticle delivery system for inhalation has been disclosed recently (WO2009121631), wherein particles were administered by endotracheal instillation to mice.
Borlak J., et al. (EP2106806), described an improved drug delivery system to the lung consisting of:                a polymer-based nanoparticle,        a maleimide-based molecular linker,        a targeting agent such as an antibody, a low molecular weight compound or a protein (preferably covalently linked to the linker),        a drug.        
Such nanoparticles had a mean size of 150 to 180 nm. According to the inventors, such delivery system took advantage of the molecular linker having a lipophilic portion which non-covalently anchors to the particle's polymeric matrix and a second portion comprising a maleimide compound to which it is possible to bind a targeting agent. Still according to the inventors, the targeting agent could be a member of a binding couple such as avidin-biotin. However, the specific embodiment involving avidin-biotin was not described in an enabling manner in this document.
Ethanol based formulations have been previously reported to be suitable for nebulisation of proteins with biological activity including enzymes (Choi W. S., et al., Proc. Natl. Acad. Sci., 2001, 98, 20, 11103). However, very limited exposure to inhaled ethanol is disclosed (i.e. 10 minutes) since longer inhalation period can provoke inflammatory side effects.
Therefore and as alluded herein-above, the physical stresses inherent to nebulisation, coupled to the formation of a large area of air-water interface, can destabilize the structure of many proteins.
[111In]-avidin and [99mTc]-biotin-liposomes administration through the pleural route has been disclosed and proved to be a better route to target lung mediastinal nodes than intraperitoneal route (Medina L. A., et al., Nucl. Med. Biol., 2004, 31, 1, 41). However, injection in the pleural cavity implies an invasive procedure and is not adequate to obtain homogenous distribution in the lung tissue. In fact, the intention of Medina's work was to target mediastinal lymph nodes. Moreover, the radiolabelled biotin needed to be formulated in liposome to improve pharmacokinetic and pharmacodynamic features.
Therefore, there is a strong medical need in providing specific way of administration to directly deliver to the diseased lung an efficacious amount of therapeutic agent in order to avoid the bottlenecks associated with:                oral delivery (e.g., drug permeability issues, first-pass effect); and/or        other systemic drug delivery routes which can be correlated to severe if not compromising toxic side effects and/or high clearance issues; and/or        instability of the therapeutic according to its administration route and/or metabolic process; and/or        multiple administrations of the therapeutic every day        
It has now been surprisingly found that The oxidized avidin, administered by inhalation, link to the surface of lung epithelial cells uniformly down to the alveoli, and that it does not surprisingly bind to the respiratory upper track (e.g., trachea).
This finding was completely unexpected since The oxidized avidin did not bind to tissue surfaces like skin, eye or bladder unless a damage was procured to said surfaces. It was also surprising that such mode of administration preserves the chemical integrity of the protein.